HIV Integrase Structural Biology

描述(申请人提供)：HIV-1整合酶(IN)与病毒DNA末端在病毒感染细胞的细胞质前整合复合体(PIC)内的分子相互作用尚不清楚。了解这些相互作用对于了解针对IN的链转移抑制剂(STI)、这些抑制剂的耐药性以及第二代抑制剂的开发至关重要。使用纯化的HIV IN和病毒DNA底物，我们已经鉴定了天然琼脂糖凝胶上的瞬时突触复合体(SC)，该复合体具有与PIC相关的特性。我们将利用这些新的进展来理解协同整合：1)利用纯化的IN单体确定HIV SC组装的功能机制；2)探索在没有或存在链转移抑制剂(STI)的情况下，以小DNA寡核苷酸(ODN)为底物产生均一的HIV IN-DNA复合体的条件；以及3)启动HIV IN-DNA复合体的共结晶研究。 最先进的机器人技术。泡沫状病毒(PFV)整合酶原型(Cherepanov组)的晶体结构极大地提高了我们对IN亚单位与同源DNA末端相互作用的理解，以促进协同整合。PFV IN主要是一种单体，允许DNA末端之间的结构域间互补形成连接体。在协调整合方面，PFV整合体和HIV SC在功能上是相同的。对HIV SC的原子分辨率研究是必要的，因为PFV和HIV IN之间的氨基酸同源性在它们各自的催化中心之外是最小的。为什么重组HIV IN不能有效地利用ODN进行协同整合，这一谜团存在了20年之久。我们最近在解开这个谜团方面取得了两项最新进展，最近发表于去年10月(生物化学。50：9788-9796,2011)。首先，我们确定我们被广泛研究的HIV-1IN是以高度溶解的单体存在的。第二，我们发现我们纯化的HIV IN使用U5 LTRODN(18bp到42bp，3‘OH凹陷或钝化)进行高效的协同整合活性，其中IN和ODN都在微摩尔浓度。最后，我们最近还确定了几个STI(&gt；200 nm)，它们使用钝端或凹端的U5底物选择性地产生新的IN-Single DNA(ISD)复合体。综上所述，我们假设HIV IN单体有助于将活性四聚体组装到病毒DNA上进行协同整合。我们将探索在没有和有STI的情况下产生可溶的HIV IN-DNA复合体的条件，用于共结晶研究。 公共卫生相关性：人类感染艾滋病毒-1通常是致命的，除非患者接受高效抗逆转录病毒药物治疗(HAART)。最近将抑制HIV-1整合酶的Raltegravir添加到联合药物治疗中，显着增加了HAART的成功。我们提出的研究将利用野生型和耐药的HIV整合酶突变体，为拉替格列韦相关的结构机制提供生化和原子解析方面的见解。

DESCRIPTION (provided by applicant): Molecular interactions of HIV-1 integrase (IN) with the viral DNA ends within the cytoplasmic preintegration complex (PIC) in virus infected cells are not clearly understood. Knowledge of these interactions is critical to understand strand transfer inhibitors (STI) directed against IN, drug-resistance to these inhibitors, and development of second generation inhibitors. Using purified HIV IN and viral DNA substrates, we have identified the transient synaptic complex (SC) on native agarose gels that shares properties associated with the PIC. We will use these new advancements for understanding concerted integration to: 1) determine the functional mechanisms of HIV SC assembly using purified IN monomers; 2) explore conditions to produce homogenous HIV IN-DNA complexes using small DNA oligonucleotides (ODN) as substrates, without or in the presence of strand transfer inhibitors (STI); and 3) initiate co-crystallization studies of HIV IN-DNA complexes using state-of-the-art robotic technologies. The crystal structure of the prototype foamy virus (PFV) intasome (Cherepanov group) has greatly enhanced our understanding of IN subunit interactions with the cognate DNA ends to promote concerted integration. PFV IN is mainly a monomer that allows interdomain complementation between DNA ends to form the intasome. The PFV intasome and the HIV SC are functionally equivalent for concerted integration. Atomic resolution studies of the HIV SC are necessary because the amino acid identity between PFV and HIV IN is minimal outside their respective catalytic centers. A mystery existed for 20 years why recombinant HIV IN was not capable of efficiently using ODN for concerted integration. We have made two recent advances towards solving this mystery recently published this last October (Biochem. 50:9788-9796, 2011). One, we determined that our extensively studied HIV-1 IN exists as a highly soluble monomer. Two, we discovered that our purified HIV IN uses U5 LTR ODN (18 bp to 42 bp, 3' OH recessed or blunt) for highly efficient concerted integration activity where both IN and ODN are in the micromolar concentrations. Finally, we also recently identified several STI (>200 nM) that selectively produce a new IN-single DNA (ISD) complex using either blunt or recessed ended U5 substrates. In summary, we hypothesize that HIV IN monomers facilitate the assembly of the active tetramer onto viral DNA for concerted integration. We will explore conditions to produce soluble HIV IN-DNA complexes, without and with STI, for co-crystallization studies. PUBLIC HEALTH RELEVANCE: Infection of humans by HIV-1 is generally lethal unless the patient is placed on highly active antiretroviral drug therapy (HAART). The recent addition of Raltegravir which inhibits the HIV-1 integrase to combinational drug therapies has significantly increased the success of HAART. Our proposed studies will provide biochemical and atomic resolution insights into the structural mechanisms associated with Raltegravir using wild type and drug-resistant HIV integrase mutants.